The goal of the research described in this proposal is an understanding of the molecular architecture of eukaryotic chromosomes. The organism used for this investigation is Drosphila melanogaster. A direct structural and biochemical approach is outlined. Central to these studies is the development of three-dimensional image reconstruction schemes using computer image enhancement and noise filtration techniques. Three-dimensional data from optical microscopy and from high voltage electron microscopy of chromosomes and nuclei, will be obtained. This image processing schema does not depend on inherent order in the sample, but, in contrast will allow determination of a structure without preconception of a given architecture. Using methodologies similar to X-ray crystalography, the three-dimensional object is analyzed in the computer and the data is built into a model, followed by refinement of the model and quantitative comparisons with other similar structures. These cellular tomographic tools will be generally useful in other cell biological studies from other laboratories. Two levels of nuclear structural organization will be studied. Three-dimensional folding patterns of interphase chromosomes in intact nuclei from diploid cells, developmentally staged embryos or polytene salivary glands will be studied. Second, high resolution investigation of nuclear interphase chromosome architecture will be undertaken. Both these levels of structure will integrate questions relating to ectopic fibers (pairing), gene activation (puffing), heterochromatin (the chromocenter) and organization of polytene bands plus interbands. Extensive use will be made of genetic manipulation involving chromosomal aberrations and the existing monoclonal antibody library to Drosophila embryo nuclear proteins, as specific probes to nuclear structures. This research will provide new tools linking nuclear structure to questions and problems of genetics and gene regulation in eukaryotic cells.